Hyaluronic acid (HA) is an acid mucopolysaccharide, a non-branched high molecular weight glycosaminoglycan consists of N-acetylglucosamine and D-glucuronic acid disaccharide repeating units, and exists in the intercellular substances of animal tissues or in the capsule of some bacteria. Hyaluronic acid is widely used in medicines, cosmetics and foods, and usually has molecular weight of 105-107 Da (Dalton).
Oligomeric hyaluronic acid refers to hyaluronic acid with molecular weight of less than 10 k Da. Researches show that molecular weight remarkably influences the activity of hyaluronic acid, and hyaluronic acids with different molecular weights may have completely reversed activities (GUO, Xueping, et al, Low-molecular-weight and Oligomeric hyaluronic acids, Chinese Journal of Biochemical Pharmaceutics, 2003, 24(3): 148-150).
Researches show that oligomeric hyaluronic acid has the activity of promoting angiogenesis in vivo as well as the activity of promoting proliferation of endothelial cells in vitro, and can promote wound healing (West D C, Kumar S. The effect of hyaluronate and its oligosaccharides on endothelial cell proliferation and monolayer integrity. Exp Cell Res. 1989, 183(1): 179-96). Oligomeric hyaluronates have the biological activity of promoting angiogenesis, promoting wound healing, and possess good application prospect in the fields of medical and pharmaceutical.
During inflammatory process, oligomeric hyaluronic acid has potent activation effect on immunocompetent cells such as dendritic cells and macrophages (Termeer C, Benedix F, Sleeman J, et al. Oligosaccharides of Hyaluronan activate dendritic cells via toll-like receptor 4. J Exp Med. 2002, 195(1):99-111).
In vitro, oligomeric hyaluronic acid can inhibit growth of mice TA3/St breast cancer cells, rat CG neuroglioma cells, human HCT clone tumor cells and human LXI lung cancer cells, and has anti-tumor functions (Ghatak S, Misra S, Toole B P. Hyaluronan constitutively regulates ErbB2 phosphorylation and signaling complex formation in carcinoma cells. J Biol Chem. 2005, 280(10): 8875-83).
In addition, hydroxyl groups, carboxylic groups and other polar groups in oligomeric hyaluronates can form hydrogen band with water molecules so as to binding a large amount of water, thereby having significant water retention effect. Thus, oligomeric hyaluronates can be used in sun protection, anti-aging and moisturizing cosmetics.
At present, the methods for the degradation of hyaluronic acid are mainly divided into three groups: physical degradation, chemical degradation, and biological degradation. Physical degradation methods can hardly degrade hyaluronic acid to 10 k Da or below. Although chemical degradation methods and enzymatic methods can prepare oligomeric hyaluronic acid, chemical degradation methods need rigorous reaction conditions (e.g., high acid and base concentrations) to reach maximum degradation degree in preparing oligomeric hyaluronic acid. At the moment, the glucosidic bonds of saccharide chain are broken, and the structure of monosaccharide (glucuronic acid and acetylglucosamine) residues is broken as well, for example, the removal of acetyl groups by hydrolysis and the breakage of 6-membered ring of monosaccharide (the manifestation is the inconsistence in infrared spectrogram in comparison with the standard spectrum of European Pharmacopoeia), which have effects on biological activity of the obtained oligomeric hyaluronic acid (GUO Xueping, et al, Low-molecular-weight and Oligomeric Hyaluronic Acid, Chinese Journal of Biochemical Pharmaceutics, 2003, 24 (3): 148-150). The oligomeric hyaluronic acids prepared by chemical degradation methods are prone to browning (China Patent Application No. 201110008110.9), and production process thereof may pollute environment. When hyaluronic acid is prepared by enzymatic methods, only glucosidic bonds between monosaccharides are broken, and other structures are not broken. In addition, enzymatic methods employ moderate reaction conditions, and do not use strong acids and strong bases. The obtained oligomeric hyaluronic acids are not prone to browning, environmental pollution is avoided, and the oligomeric hyaluronic acids as prepared by enzymatic methods have integral structure and the infrared spectrum thereof is consistent with the standard spectrum of European Pharmacopoeia. Thus, enzymatic methods are most suitable for preparing oligomeric hyaluronic acid.
Enzymes used for degrading hyaluronic acid are mainly hyaluronidases, which can be divided into 3 groups according to their mechanisms: (1) endo-β-N-acetylglucosaminidase, a hydrolase and acts on) β-1,4-glucosidic bond, the main products are tetrasaccharides, can act on chondroitin or chondroitin sulfate and has transglycosidase activity; (2) hyaluronidase derived from hirudo or hookworm, it is endo-β-glucuronidase, acts on β-1,3-glucosidic bond, is also a hydrolase, the main degradation products are tetrasaccharides, can specifically degrade hyaluronic acid; (3) bacterial hyaluronidase, it is also called as hyaluronate lyase, acts on β-1,4-glucosidic bond, and produces 4,5-unsaturated disaccharides via β-elimination mechanism (Kreil, G, Hyaluronidases—a group of neglected enzymes, Protein Sci, 1995, 4(9): 1666-1669).
At present, methods for industrial production of oligomeric hyaluronic acid or salts thereof are chemical degradation methods. Since the sources of animal tissues containing hyaluronidase are limited, some reports show that microbe-sourced hyaluronidase fermentation broths have lower enzyme activity per unit, and the maximum enzyme activity of fermentation broths is 1.3×102 IU/mL (W02010130810A1; Eileen Ingham, K. T. Holland, G. Gowland and W. J. Cunliffe, Purification and Partial Characterization of Hyaluronate Lyase (EC4.2.2.1) from Propionibacterium acnes, Journal of General Microbiology (1979), 115, 411-418), other reports all show the enzyme activity of lower than 100 IU/mL, and hyaluronidase cannot be produced in large scale, and thus oligomeric hyaluronic acid or salts thereof cannot be produced in large scale by enzymatic methods.
In addition, hyaluronic acid with molecular weight of 104 Da-106 Da is usually called as low-molecular-weight hyaluronic acid (LMW-HA). LMW-HA with molecular weight of 20,000-60,000 can stimulate the proliferation of bone cells cultured in vitro, increase the number of bone cell colonies and the area of single colony in cultural media (U.S. Pat. No. 5,646,129). By using eye drops containing tobramycin and HA with molecular weight of 500,000 in treatment of bacterial keratohelcosis, healing time was significantly shortened in comparison with eye drops containing tobramycin only (Gandolfi S A, Massari A, Orsoni J G. Low-molecular-weight sodium hyaluronate in the treatment of bacterial corneal ulcers s[J]. Graefes Arch Clin Exp Ophthalmol, 1992, 230(1): 20-23). Korea patent (KR20080087941) provides a composition of HA with molecular weight of less than 100,000, the composition is prone to absorption in human body, and oral administration thereof can effectively remove skin wrinkle and improve skin elasticity. Japanese patent (JP2000-103723) provides a composition containing HA with molecular weight of 200,000-400,000, the composition can promote hair growth. However, similar to oligomeric hyaluronic acid or salts thereof, LMW-HA or salts thereof as prepared by enzymatic degradation methods are more integral in structure than those prepared by chemical methods, and LMW-HA or salts thereof as prepared by chemical methods may have ring opening and deacetylation in their structure.
LMW-HA has smaller relative molecular weight, and thus can be well absorbed by skin in external use, promote skin nutrient supply and waste excretion, so as to prevent skin aging, achieve deep moisturizing and maintain beauty, promote proliferation and differentiation of epithelial cells, scavenge free radicals, as well as repair skin injury caused by sun light or ultraviolet light. Oral LMW-HA is prone to absorption, can activate skin cells, keep skin moisture and meanwhile provide well immune-enhancement function and anti-aging function. Thus, the HA used in cosmetics and health foods are usually LMW-HA. In addition, LMW-HA can also promote angiogenesis, cell immunity activation and osteogenesis, has good therapeutic effects in treatment of bacterial keratohelcosis, and thus is widely used in the field of medicine.
LMW-HA is obtained by degrading high molecular weight HA, and degradation methods are mainly 3 groups, i.e., physical degradation, chemical degradation, and biological degradation. Although ultrasonic degradation method as physical degradation method is free of chemical agents and enzymes, it can hardly degrade high molecular weight HA to reach molecular weight of 200 k Da or below (QIN Caifeng, WANG Miao, CHEN Xiaofeng, Studying of Degradation methods and Process Conditions of Hyaluronic Acid, [J]. 2007, 4: 32-36). Mechanical grinding method is also an effective method to reduce relative molecular weight of HA. A patent discloses grinding time at room temperature is 0.5 h-12 h, oscillation frequency is 10 Hz-30 Hz, but it is not suitable for production in large scale (CN101942037A). Chemical degradation method may break structure, which may break not only glucosidic bonds of saccharide chain, but also structure of monosaccharide (glucuronic acid and acetylglucosamine) residues, for example, acetyl groups are removed by hydrolysis and 6-membered ring of monosaccharide is broken.
Some reports show a process for preparing low-molecular-weight HA by enzymatic degradation method, in which HA is firstly isolated and purified from fermentation broth, then hyaluronidase is added for enzymatic degradation, following membrane filtration, ethanol precipitation, dehyarating and drying to obtain low-molecular-weight HA (CN101020724A), the hyaluronidase used in the process is a finished product of hyaluronidase, which is very expensive and can hardly be used for industrial production. At present, some reports show microbe-sourced hyaluronidase fermentation broths have lower enzyme activity per unit, the maximum enzyme activity of fermentation broths is 1.3×102 IU/mL (W02010130810A1), other reports all show enzyme activity of lower than 100 IU/mL, therefore, at present, low-molecular-weight HA can hardly be produced in large scale by enzymatic methods.